1. Field of the Invention
This invention relates to a measuring method and apparatus using attenuation in total internal reflection such as a surface plasmon sensor for analyzing a sample on the basis of generation of surface plasmon.
2. Description of the Related Art
In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon.
There have been proposed various surface plasmon sensors for quantitatively analyzing a material in a sample utilizing a phenomenon that such surface plasmon is excited by light waves. Among those, one employing a system called xe2x80x9cKretschmann configurationxe2x80x9d is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism, a metal film which is formed on one face of the dielectric block and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of surface plasmon resonance, i.e., a state of attenuation in total internal reflection.
In order to obtain various angles of incidence of the light beam to the interface, a relatively thin incident light beam may be caused to impinge upon the interface while deflecting the incident light beam or a relatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light so that components of the incident light beam impinge upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle which varies as the incident light beam is deflected may be detected by a photodetector which is moved in synchronization with deflection of the incident light beam or by an area sensor extending in the direction in which reflected light beam is moved as a result of deflection. In the latter case, an area sensor which extends in directions so that all the components of light reflected from the interface at various angles can be detected by the area sensor may be used.
In such a plasmon resonance sensor, when a light beam impinges upon the interface at a particular angle of incidence xcex8sp not smaller than the angle of total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample. When the wave number vector of the evanescent waves is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector. The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the light beam to impinge upon the interface in the form of p-polarized light.
When the wave number of the surface plasmon can be known from the angle of incidence xcex8sp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is             K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ϵ              m                        ⁡                          (              ω              )                                ⁢                      ϵ            s                                                              ϵ              m                        ⁡                          (              ω              )                                +                      ϵ            s                              
wherein Ksp represents the wave number of the surface plasmon, xcfx89 represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and ∈m and ∈s respectively represent the dielectric constants of the metal and the sample.
A property related to the dielectric constant ∈s (refractive index) of the sample can be detected by detecting the angle of incidence xcex8sp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops (this angel xcex8sp will be referred to as xe2x80x9cthe attenuation angle xcex8spxe2x80x9d, hereinbelow).
In such a surface plasmon sensor, it has been proposed, in order to measure the attenuation angle xcex8sp accurately with a wide dynamic range, to use a photodetector in the form of an array of a plurality of photodetector elements arranged in a predetermined direction so that light beam components reflected at different angles at the interface impinge upon different photodetector elements as disclosed in Japanese Unexamined Patent Publication No. 11 (1999)-326194.
In this case, the output signals output from the photodetector elements are generally differentiated in the direction in which the photodetector elements are arranged, and the refractive-index-related property of the material to be measured is generally obtained on the basis of the differentials.
As a similar apparatus utilizing the phenomenon of attenuation in total internal reflection (ATR), there has been known a leaky mode sensor described in, for instance, xe2x80x9cSpectral Researchxe2x80x9d Vol.47, No.1 (1998), pp21 to 23 and pp26 and 27. The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one face of the dielectric block, an optical waveguide layer which is formed on the clad layer and is brought into contact with a sample liquid, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block to impinge upon the interface of the dielectric block and the metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of waveguide mode excitation, i.e., a state of attenuation in total internal reflection (ATR).
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, only light having a particular wave number and impinging upon the optical waveguide layer at a particular angle of incidence comes to propagate through the optical waveguide layer in a waveguide mode after passing through the clad layer. When the waveguide mode is thus excited, almost all the incident light is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer in a waveguide mode depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the angle of incidence at which the attenuation in total internal reflection occurs.
Also in such a leaky mode sensor, a photodetector in the form of an array of a plurality of photodetector elements can be used to detect the position of the dark line generated due to the attenuation in total internal reflection, and at the same time differentiation of the output signals output from the photodetector elements is often applied.
The surface plasmon sensor and the leaky mode sensor are sometimes used in random screening for finding a specific material combined with a predetermined sensing material in the field of pharmacy. In this case, a sensing material is fixed on the film layer (the metal film in the case of the surface plasmon sensor and the clad layer and the optical waveguide layer in the case of the leaky mode sensor), and a sample liquid containing a material to be analyzed is spotted on the sensing material. Then the attenuation angle xcex8sp is repeatedly measured each time a predetermined time lapses.
When the sample material (the material to be analyzed in the sample liquid) is combined with the sensing material, the refractive index of the sensing material changes with time due to combination with the sample material. Accordingly, by measuring the attenuation angle xcex8sp, at which attenuation in total internal reflection takes place, for every predetermined time, thereby detecting whether the attenuation angle xcex8sp changes (to know the state of combination of the sample material with the sensing material), it is possible to know whether the sample material is a specific material to be combined with the sensing material. As combinations of such a specific material and a sensing material, there have been known combinations of an antigen and an antibody and of an antibody and another antibody. For example, rabbit antihuman IgG antibody is fixed on the surface of the film layer as the sensing material with human IgG antibody employed as the specific material.
In order to detect the state of combination of the sample material with the sensing material, the total reflection attenuation angle xcex8sp (the angle of incidence xcex8sp at which attenuation in total internal reflection takes place) itself need not necessarily be detected. For example, the amount of change in the total reflection attenuation angle xcex8sp after the sample liquid is spotted onto the sensing material is measured and the state of combination of the sample material with the sensing material may be measured on the basis of the amount of change of the total reflection attenuation angle xcex8sp. When a photodetector in the form of an array of a plurality of photodetector elements and differentiation of the output signals output from the photodetector elements are employed, the state of combination of the sample material with the sensing material can be measured on the basis of the amount of change of the differentiation of the output signals. (See our Japanese Patent Application 2000-398309.)
In the practical apparatuses utilizing the phenomenon of attenuation in total internal reflection such as a surface plasmon sensor or a leaky mode sensor, the amount of change of the total reflection attenuation angle xcex8sp is measured by spotting a sample liquid comprising solvent and a sample material onto a film layer formed on the bottom of a measuring chip in the form of a cup or dish.
When a sample liquid is spotted on a measuring chip and a sample material in the sample liquid is combined with the sensing material, the refractive index of the sensing material changes and the attenuation angle xcex8sp changes. However, strictly speaking, the change of the attenuation angle xcex8sp does not solely depend upon the state of combination of the sample material with the sensing material but also depends upon action between the sensing material and the solvent of the sample liquid and difference in sensitivity among measuring apparatuses.
That is, even when a false sample liquid which solely consists of solvent and includes no sample material is supplied to a sensing material, the attenuation angle xcex8sp slightly changes as shown by the solid line in FIG. 4. The change of the attenuation angle xcex8sp produces a measuring error. In order to avoid production of this measuring error, we, this applicant has proposed, in our Japanese Patent Application 2001-049681, a method of judging whether a sensing material is combined with a sample material, in which a corrected attenuation angle change is obtained by subtracting the change of the attenuation angle xcex8sp in a reference chip supplied with a false sample liquid from the change of the attenuation angle xcex8sp in a measuring chip supplied with a real sample liquid and whether the sensing material is combined with a sample material in the sample liquid is judged on the basis of the corrected attenuation angle change. In accordance with the method, since the corrected attenuation angle change becomes substantially 0 when the sensing material is not combined with the sample material, whether the sensing material is combined with a sample material in the sample liquid can be easily judged.
However, this method is disadvantageous in that since the corrected attenuation angle change does not become completely 0 even if the sensing material is not combined with the sample material, this method is not effective when the sample material is small in molecular weight though effective when the sample material is large in molecular weight.
The attenuation angle change in a measuring chip supplied with a false sample liquid as measured by these inventors is as shown by the solid line in FIG. 4, whereas the attenuation angle change in a reference chip supplied with the same false sample liquid as measured by these inventors is as shown by the broken line in FIG. 4. The corrected attenuation angle change in this case is as shown by the chained line in FIG. 4. Since the sensing material is combined with the sample material in neither cases, the corrected attenuation angle should be 0. However the attenuation angle change after one hour shown by the chained line shows a value of 900 in terms of molecular weight. That is, the corrected attenuation angle, which should be 0, is too large, which deteriorates the measuring accuracy.
A reason why there is produced a difference between the attenuation angle change in a reference chip and that in a measuring chip supplied with the same false sample liquid is, for instance, the difference in thickness of the metal films between the measuring chip and the reference chip. Another reason may be a difference in sensitivity between the photodetector means of the measuring unit and that of the reference unit.
Further, this applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which a plurality of measuring chips are placed on, for instance, a turn table and are measured in sequence, thereby shortening the time required to measure a lot of samples. (Japanese Unexamined Patent Publication No. 2001-330560)
Further, this applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which measuring chips each having a plurality of sample liquid holding portions are used so that measurement on a plurality of samples can be done at one time without moving the measuring chips. (Japanese Patent Application 2001-397411)
In view of the foregoing observations and description, the primary object of the present invention is to provide a measuring method and apparatus using attenuation in total internal reflection which can suppress the difference in sensitivity between the photodetector means of the measuring unit and that of the reference unit and can more accurately detect the state of attenuation in total internal reflection.
Another object of the present invention is to provide a measuring method and apparatus which can suppress the difference in sensitivity among measuring apparatuses and can more accurately analyze the sample.
In accordance with a first aspect of the present invention, there is provided a measuring method for detecting a state of attenuation in total internal reflection by the use of a measuring apparatus comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a first film layer formed on one face of the first dielectric block and a first sample liquid holding system which holds a sample liquid on the surface of the first film layer, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the first film layer; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the first film layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the first film layer,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a second film layer formed on one face of the second dielectric block and a second sample liquid holding system which holds a false sample liquid on the surface of the second film layer, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the second film layer; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the first film layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the second film layer, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises the steps of
detecting the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
calibrating result of measurement by the measuring means on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In accordance with a second aspect of the present invention, the method of the present invention is applied to a surface plasmon resonance sensor. That is, in accordance with the second aspect of the present invention, there is provided a measuring method for detecting a state of attenuation in total internal reflection by the use of a measuring apparatus comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a first metal film formed on one face of the first dielectric block and a first sample liquid holding system which holds a sample liquid on the surface of the first metal film, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the first metal film; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the first metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the first metal film,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a second metal film formed on one face of the second dielectric block and a second sample liquid holding system which holds a false sample liquid on the surface of the second metal film, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the second metal film; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the first metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the second metal film, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection due to generation of surface plasmon resonance on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises the steps of
detecting the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
calibrating result of measurement by the measuring means on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In accordance with a third aspect of the present invention, the method of the present invention is applied to a leaky mode sensor. That is, in accordance with the third aspect of the present invention, there is provided a measuring method for detecting a state of attenuation in total internal reflection by the use of a measuring apparatus comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a clad layer formed on one face of the first dielectric block, an optical waveguide layer which is formed on the clad layer and a first sample liquid holding system which holds a sample liquid on the surface of the clad layer, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the clad layer; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the clad layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the clad layer,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a clad layer formed on one face of the second dielectric block, an optical waveguide layer which is formed on the clad layer and a second sample liquid holding system which holds a false sample liquid on the surface of the clad layer, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the clad layer; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the clad layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the clad layer, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection due to excitation of waveguide mode on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises the steps of
detecting the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
calibrating result of measurement by the measuring means on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In the methods in accordance with the first to third aspects of the present invention, the xe2x80x9cdifference in sensitivity between the measuring unit and the reference unitxe2x80x9d may be detected, for instance, by causing the first and second light beams to impinge upon the interfaces at various angles of incidence so that total internal reflection conditions are satisfied at the interfaces with false sample liquids held by the sample holding liquid systems of the respective units, detecting changes of the attenuation in total internal reflection on the basis of the intensities of the light beams reflected at the interfaces by the measuring unit and the reference unit and comparing results of detection by the measuring unit and the reference unit.
In accordance with fourth to sixth aspects of the present invention, there are provided measuring apparatuses respectively for carrying out the measuring methods in accordance with the first to third aspects of the present invention.
That is, in accordance with the fourth aspect of the present invention, there is provided a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a first film layer formed on one face of the first dielectric block and a first sample liquid holding system which holds a sample liquid on the surface of the first film layer, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the first film layer; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the first film layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the first film layer,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a second film layer formed on one face of the second dielectric block and a second sample liquid holding system which holds a false sample liquid on the surface of the second film layer, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the second film layer; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the first film layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the second film layer, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises that
there is further provided a sensitivity difference detecting means which detects the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
said measuring means calibrates result of measurement on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In accordance with the fifth aspect of the present invention, there is provided a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a first metal film formed on one face of the first dielectric block and a first sample liquid holding system which holds a sample liquid on the surface of the first metal film, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the first metal film; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the first metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the first metal film,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a second metal film formed on one face of the second dielectric block and a second sample liquid holding system which holds a false sample liquid on the surface of the second metal film, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the second metal film; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the first metal film at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the second metal film, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection due to generation of surface plasmon resonance on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises that
there is further provided a sensitivity difference detecting means which detects the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
said measuring means calibrates result of measurement on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In accordance with the sixth aspect of the present invention, there is provided a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection comprising
a measuring unit comprising a first light source emitting a first light beam; a measuring chip consisting of a first dielectric block transparent to the first light beam, a clad layer formed on one face of the first dielectric block, an optical waveguide layer which is formed on the clad layer and a first sample liquid holding system which holds a sample liquid on the surface of the clad layer, the sample liquid comprising solvent and a sample; a sensing material which can be combined with a specific material in the sample liquid and is disposed on the clad layer; a first optical system which causes the first light beam to enter the first dielectric block to impinge upon the interface of the first dielectric block and the clad layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a first photodetector means which detects the intensity of the first light beam reflected in total internal reflection at the interface of the first dielectric block and the clad layer,
a reference unit comprising a second light source emitting a second light beam; a reference chip consisting of a second dielectric block transparent to the second light beam, a clad layer formed on one face of the second dielectric block, an optical waveguide layer which is formed on the clad layer and a second sample liquid holding system which holds a false sample liquid on the surface of the clad layer, the false sample liquid comprising said solvent; a sensing material which can be combined with said specific material in the sample liquid and is disposed on the clad layer; a second optical system which causes the second light beam to enter the second dielectric block to impinge upon the interface of the second dielectric block and the clad layer at various angles of incidence so that total internal reflection conditions are satisfied at the interface; and a second photodetector means which detects the intensity of the second light beam reflected in total internal reflection at the interface of the second dielectric block and the clad layer, and
a measuring means which corrects result of detection by the first photodetector means on the basis of result of detection by the second photodetector means and measures the change of a state of attenuation in total internal reflection due to excitation of waveguide mode on the basis of the corrected result of detection by the first photodetector means,
wherein the improvement comprises that
there is further provided a sensitivity difference detecting means which detects the difference in sensitivity between the measuring unit and the reference unit before initiating the measurement of the change of a state of attenuation in total internal reflection, and
said measuring means calibrates result of measurement on the basis of the difference in sensitivity between the measuring unit and the reference unit.
In the measuring apparatuses in accordance with the fourth to sixth aspects of the present invention, the sensitivity difference detecting means may be a means for detecting the xe2x80x9cdifference in sensitivity between the measuring unit and the reference unitxe2x80x9d, for instance, by causing the first and second light beams to impinge upon the interfaces at various angles of incidence so that total internal reflection conditions are satisfied at the interfaces with false sample liquids held by the sample holding liquid systems of the respective units, detecting changes of the attenuation in total internal reflection on the basis of the intensities of the light beams reflected at the interfaces by the measuring unit and the reference unit and comparing results of detection by the measuring unit and the reference unit.
Though it is preferred that the xe2x80x9cfalse sample liquidxe2x80x9d solely comprises the same solvent used in the sample liquid, but need not be so. For example, a liquid which is substantially the same in characteristics of action with the sensing material and/or the optical characteristics as the solvent used in the sample liquid may be used as the false sample liquid.
The sensing material and the specific material in the sample liquid may be combined each other, for instance, by interaction between proteins, interaction between DNA and protein, interaction between sugar and protein, interaction between protein and peptide, interaction between fat and protein or chemical reactions.
The first and second light sources may be either separate light sources or a single light source common to the measuring unit and the reference unit. The first and second optical systems may be either separate optical systems or a single optical system common to the measuring unit and the reference unit. The first and second photodetector means may be either separate photodetector means or a single photodetector means common to the measuring unit and the reference unit.
In accordance with the measuring methods and the measuring apparatuses in accordance with the first to sixth aspects of the present invention, since the difference in sensitivity between the measuring unit and the reference unit is detected before initiating the measurement of the change of a state of attenuation in total internal reflection, and result of measurement by the measuring means is calibrated on the basis of the difference in sensitivity between the measuring unit and the reference unit, influence of the difference in sensitivity between the measuring unit and the reference unit on the measuring accuracy can be suppressed and the accuracy in measurement of the change of a state of attenuation in total internal reflection can be improved.
When the xe2x80x9cdifference in sensitivity between the measuring unit and the reference unitxe2x80x9d is detected by causing the first and second light beams to impinge upon the interfaces at various angles of incidence so that total internal reflection conditions are satisfied at the interfaces with false sample liquids held by the sample holding liquid systems of the respective units, detecting changes of the state of the attenuation in total internal reflection on the basis of the intensities of the light beams reflected at the interfaces by the measuring unit and the reference unit and comparing results of detection by the measuring unit and the reference unit, influence of the sensitivity difference between the measuring chip and the reference chip and/or the sensitivity difference between the photodetector means of the measuring unit and the reference unit can be suppressed and the accuracy in measuring the state of combination between the sensing material and the sample can be improved.
In accordance with a seventh aspect of the present invention, there is provided a measuring method for analyzing a sample by the use of a measuring apparatus comprising; a measuring chip consisting of a dielectric block, a film layer formed on one face of the dielectric block and a sample holding system which holds the sample on the surface of the film layer; a light source which emits a light beam; an optical system which causes the light beam to enter the dielectric block at an angle of incidence such that total internal reflection conditions are satisfied at the interface of the dielectric block and the film layer; and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface, wherein the improvement comprises the steps of
measuring the refractive indexes of a plurality of reference samples whose refractive indexes are known, thereby obtaining a calibration standard on the basis of result of measurement on the reference samples, and calibrating result of measurement on the sample on the basis of the calibration standard.
In accordance with an eighth aspect of the present invention, there is provided a measuring apparatus comprising; a measuring chip consisting of a dielectric block, a film layer formed on one face of the dielectric block and a sample holding system which holds the sample on the surface of the film layer; a light source which emits a light beam; an optical system which causes the light beam to enter the dielectric block at an angle of incidence such that total internal reflection conditions are satisfied at the interface of the dielectric block and the film layer; and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface, wherein the improvement comprises
a calibrating means which obtains a calibration standard by measuring the refractive indexes of a plurality of reference samples whose refractive indexes are known and calibrates result of measurement on the sample on the basis of the calibration standard.
The seventh and eighth aspects of the present invention can be applied to, for instance, a surface plasmon resonance sensor where the film layer is a metal film and a leaky mode sensor where the film layer comprises a clad layer formed on one face of the dielectric block and an optical waveguide layer formed on the clad layer.
In the measuring apparatuses of the seventh and eighth aspects of the present invention, the sample may be analyzed in various ways on the basis of the intensity of the light beam reflected in total internal reflection at the interface. For example, the sample may be analyzed by detecting the position (angle) of a dark line generated by attenuation in total internal reflection by causing the light beam to impinge upon the interface so that total internal reflection conditions are satisfied at the interface and various angles of incidence of the light beam to the interface are obtained, and measuring the intensity of the light beam reflected in total internal reflection at the interface by positions corresponding to angles of incidence, or by causing a plurality of light beams having different wavelengths to impinge upon the interface so that total internal reflection conditions are satisfied at the interface, measuring the intensities of the light beams reflected in total internal reflection at the interface by wavelengths and detecting the degree of attenuation in total internal reflection by wavelengths as disclosed in xe2x80x9cPorous Gold in Surface Plasmon Resonance Measurementxe2x80x9d by D. V. Noort, K. Johansen, and C. F. Mandenius (EUROSENSORS XIII, 1999, pp.585-588).
In the seventh and eighth aspects of the present invention, it is preferred that the measuring apparatus further comprises a reference sample generating means which mixes two samples, whose refractive indexes (dielectric constants) are known, in different proportions to generate a plurality of reference samples whose refractive indexes are between the refractive indexes of said two samples whose refractive indexes are known.
The reference samples need not be liquid but may be, for instance, solid. For example, the xe2x80x9crefractive indexes of a plurality of reference samples whose refractive indexes are knownxe2x80x9d may be obtained by the use of a measuring chip (a calibration jig) where a solid material of a known refractive index (dielectric constant) is fixed (e.g., by deposition) on the film layer.
In this case, it is preferred that the calibration jig comprises a plurality of measuring chips which are arranged in a row and integrated with each other into a measuring chip unit and a plurality of solid materials whose refractive indexes are known and which are respectively fixed on the film layers of the measuring chips. In such a measuring chip unit, the dielectric blocks of the respective measuring chips may be formed integrally.
Further, it is preferred that the photodetector means comprises a plurality of photodetector elements which are arranged in a predetermined direction to receive the light beam reflected at the interface in total internal reflection, a differential means which differentiates detecting signals output from the photodetector elements in the direction in which the photodetector elements are arranged and outputs the differentials obtained, and a measuring means which subtracts from a differential, close to a point at which change of the detecting signal in the direction in which the photodetector elements are arranged changes from decrease to increase, the initial value of the differential and measures the change with time of the differential.
Though, it is preferred that the xe2x80x9cdifferential close to a point at which change of the detecting signal in the direction in which the photodetector elements are arranged changes from decrease to increasexe2x80x9d be the differential closest to the point, it need not be necessarily so but may be any one close to the point. Further, the xe2x80x9cinitial value of the differentialxe2x80x9d may be either a differential which is obtained for the first time after measurement is initiated and is close to a point at which change of the detecting signal in the direction in which the photodetector elements are arranged changes from decrease to increase, or a value set as an initial value by carrying out an operation such as feedback for shifting a measured value at the beginning of measurement to a value close to 0. The measuring means may subtract xe2x80x9cfrom a differential, close to a point at which change of the detecting signal in the direction in which the photodetector elements are arranged changes from decrease to increase, the initial value of the differentialxe2x80x9d, for instance, by the use of a subtracter or a differential circuit.
In accordance with the seventh and eighth embodiments of the present invention, since the refractive indexes of a plurality of reference samples whose refractive indexes are known are measured, thereby obtaining a calibration standard on the basis of result of measurement on the reference samples, and result of measurement on the sample is calibrated on the basis of the calibration standard, the difference in sensitivity among measuring apparatuses can be suppressed and the sample can be analyzed more accurately.